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Journal of PathologyJ Pathol 2016; 240: 3–14Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/path.4745

ORIGINAL PAPER

The Hippo effector TAZ (WWTR1) transforms myoblasts and TAZabundance is associated with reduced survival in embryonalrhabdomyosarcomaAbdalla Mohamed,1 Congshan Sun,2 Vanessa De Mello,1 Joanna Selfe,3 Edoardo Missiaglia,4 Janet Shipley,3Graeme I Murray,1 Pete S Zammit2 and Henning Wackerhage1,*

1 School of Medicine, Dentistry and Nutrition, University of Aberdeen, UK2 Randall Division of Cell and Molecular Biophysics, King’s College London, UK3 Sarcoma Molecular Pathology Team, Divisions of Molecular Pathology and Cancer Therapeutics, Institute of Cancer Research, London, UK4 Institut Universitaire de Pathologie de Lausanne IUP, Switzerland

*Correspondence to: H Wackerhage, Technical University of Munich, Germany. E-mail: [email protected]

AbstractThe Hippo effector YAP has recently been identified as a potent driver of embryonal rhabdomyosarcoma (ERMS).Most reports suggest that the YAP paralogue TAZ (gene symbol WWTR1) functions as YAP but, in skeletalmuscle, TAZ has been reported to promote myogenic differentiation, whereas YAP inhibits it. Here, we investigatedwhether TAZ is also a rhabdomyosarcoma oncogene or whether TAZ acts as a YAP antagonist. Immunostainingof rhabdomyosarcoma tissue microarrays revealed that TAZ is significantly associated with poor survival inERMS. In 12% of fusion gene-negative rhabdomyosarcomas, the TAZ locus is gained, which is correlated withincreased expression. Constitutively active TAZ S89A significantly increased proliferation of C2C12 myoblasts and,importantly, colony formation on soft agar, suggesting transformation. However, TAZ then switches to enhancemyogenic differentiation in C2C12 myoblasts, unlike YAP. Conversely, lentiviral shRNA-mediated TAZ knockdownin human ERMS cells reduced proliferation and anchorage-independent growth. While TAZ S89A or YAP1 S127Asimilarly activated the 8XGTIIC–Luc Hippo reporter, only YAP1 S127A activated the Brachyury (T-box) reporter.Consistent with its oncogene function, TAZ S89A induced expression of the ERMS cancer stem cell gene Myf5and the serine biosynthesis pathway (Phgdh, Psat1, Psph) in C2C12 myoblasts. Thus, TAZ is associated with poorsurvival in ERMS and could act as an oncogene in rhabdomyosarcoma.© 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britainand Ireland.

Keywords: Hippo pathway; TAZ; WWTR1; embryonal rhabdomyosarcoma; myoblasts

Received 27 December 2015; Revised 4 April 2016; Accepted 27 April 2016

No conflicts of interest were declared.

Introduction

Yap (gene symbol Yap1) and Taz (gene symbol Wwtr1)are transcriptional co-factors that regulate gene expres-sion mainly by binding Tead1–4 [1,2]. Yap and Taz arenot only regulated by the Hippo pathway [3] but are alsoadditionally targeted by a network of other signallingmolecules, including PIK3CA [4], KRAS [5–7] andCTNNB1 (encoding β-catenin) [8]. Importantly, thesecrosstalking genes are affected by recurrent pan-cancermutations [9], including in embryonal rhabdomyosar-coma (ERMS) [10].

Persistent Yap hyperactivity, as a consequence ofexpressing a constitutively active YAP1 S127A mutant,results in tumours of the liver [11] and skin in mice [12].Constitutive Yap hyperactivity in activated muscle stem(satellite) cells causes ERMS-like tumours withhigh penetrance in mice [13]. Whilst no YAP1 orWWTR1 point mutations have been reported for ERMS,

mutations of several cancer genes that can crosstalk/interact with YAP or TAZ have been identified insequencing studies [10]. In addition, we and oth-ers have reported YAP1 copy number gains in somerhabdomyosarcomas [13,14].

Yap and Taz both have WW domains and can bind allTead transcription factors [1,2], but differ significantlyin their function. This is especially evident in knockoutmice, as a Yap1 knockout is embryonal lethal at E8.5[15], whereas some Wwtr1 knockout mice are bornbut later develop glomerulocystic kidney disease [16].Nonetheless, TAZ, like YAP, has been associated withcancer [17,18], suggesting that both YAP1 and WWTR1can act as oncogenes.

In the skeletal muscle lineage, high levels of YAPactivity in muscle fibres cause myopathy [19], butmore moderate increases induce skeletal muscle fibrehypertrophy [20,21]. In myoblasts and satellite cells,active Yap potently promotes proliferation [22,23] and

© 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in anymedium, provided the original work is properly cited.

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persistent YAP hyperactivity transforms satellite cellsto cause ERMS [13]. In contrast to Yap, active Tazhas been reported to promote myogenic differentia-tion [24]. The promotion of myogenic differentiationwould be anti-tumourigenic, because myoblasts withina rhabdomyosarcoma tumour fail to differentiate intopost-mitotic myocytes/myotubes [25]. This might sug-gest context-dependent function of TAZ as either anoncogene or tumour suppressor. Similarly, whilst YAPand TAZ generally function as oncogenes [17,18], it hasbeen reported that YAP can function as a tumour sup-pressor in the intestine [26], although there is no con-sensus on this [27].

The aim of this study was to test whether TAZ abun-dance is associated with survival in rhabdomyosarcomaand to characterize the cancer-specific functions of TAZin myoblasts and human ERMS cells, to identify TAZas either an oncogene and YAP agonist or as a tumoursuppressor.

Materials and methods

Human rhabdomyosarcoma tissue microarraysFor the rhabdomyosarcoma tissue microarrays,formalin-fixed, paraffin-embedded diagnostic tumourmaterial from 79 patients with RMS was collectedfrom UK centres through the Children’s Cancer andLeukaemia Group (Local Research Ethics Commit-tee Protocol Nos. 1836 and 2015 and Multi-RegionalResearch Ethics Committee 06/4/71, with consent whererequired). The histology of cases was confirmed, byreview according to World Health Organization guide-lines, to be 25 alveolar and 54 embryonal. Cores of0.6 mm diameter from three or more defined regions oftumour blocks were used to construct a tissue microar-ray [28]. A previously described tissue microarray wasalso used, containing material from 60 alveolar and171 embryonal cases [29]. Immunohistochemistry andassessment of the arrays is reported in Supplementarymaterials and methods (see supplementary material).

Cell cultureMouse C2C12 and human RD and RH30 cells werecultured in Dulbecco’s minimum essential medium(DMEM; Sigma), supplemented with 10% fetal calfserum (FCS; Hyclone). To induce differentiation, cellswere cultured in growth medium until confluence, thenthe medium was switched to DMEM with 2% horseserum (Hyclone). Human cells [30] were culturedin skeletal muscle cell growth medium (Promocell,C-23160) and passaged when needed.

Retroviral and lentiviral expression vectorsand transduction methodsWild-type TAZ and TAZ S89A cDNA were subclonedinto a pMSCV–IRES–eGFP [31] retroviral expres-sion backbone from plasmid DNA (Addgene Plasmids

24809 and 24815, deposited by Dr Jeff Wrana), creatingpMSCV–3x Flag TAZ–IRES–eGFP and pMSCV–3xFlag–TAZ S89A–IRES–eGFP constructs. Empty vec-tor pMSCV–IRES–eGFP was used as control vector.Retroviruses [23] and lentiviruses [32] were packagedin HEK293-T cells, as described previously. Cells weretransduced by incubation in diluted viral supernatant(1:4) until assayed.

ImmunocytochemistryFixed cells were permeabilized with 0.5% v/vTween-20/PBS for 6 min and blocked with 20%v/v goat serum/PBS for 30 min. The cells were incu-bated overnight at 4 ∘C with the primary antibodiesmouse anti-MyHC (DSHB, clone MF20; 1:400), mouseanti-Ki67 (Cell Signaling, 9449; 1:400) and chickenanti-GFP (Abcam, ab13970; 1:1000). Species-specific,fluorochrome-conjugated secondary antibodies wereapplied for 90 min at room temperature and the slideswere mounted with Vectashield medium with DAPI(Vector laboratories). Proliferation of RD cells wasassessed by EdU (5-ethynyl-2′-deoxyuridine) incor-poration, using 10 μM EdU for 4 h in growth medium.The Click-iT® EdU kit (Invitrogen) was used to detectincorporated EdU, following the manufacturer’s instruc-tions. Quantification of Ki67- and EdU-positive cellpercentages and myogenic fusion index is reported inSupplementary materials and methods (see supplemen-tary material).

Western blottingCells were lysed in modified RIPA buffer supple-mented with protease cocktail and phosphataseinhibitors (Sigma). Whole-cell lysates were sepa-rated by SDS–PAGE electrophoresis and transferredto a nitrocellulose membrane. The membranes werethen probed with mouse anti-Yap/Taz (Santa Cruz,101199; 1:100), rabbit anti-phospho Yap Ser127 (CellSignalling, 9411; 1:1000) or rabbit anti-Taz (Sigma,HPA007415; 1:1000). Primary antibodies were thenvisualized using species-specific conjugated secondaryantibodies (Invitrogen) and digitally imaged.

Anchorage-independent agarose transformationassayCells (1× 104) were added to 2 ml growth medium with0.5% agarose (Promega) and layered onto 2 ml 0.35%agarose-supplemented medium in six-well plates. Thecells were fed with 1 ml growth medium weekly for 4–6weeks, after which colonies were fixed with 10% aceticacid/10% methanol for 10 min, followed by stainingwith 0.005% crystal violet for 1 h, and counted usinga light microscope. Colony numbers and sizes weredetermined using ImageJ v. 1.43 (NIH, USA).

RNA extraction and reverse transcription (RT)–qPCRTotal RNA was isolated from cultured cells using Trizolreagent (Invitrogen). cDNA was synthesized by reverse

© 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd J Pathol 2016; 240: 3–14on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org www.thejournalofpathology.com

TAZ in ERMS 5

transcription, using Superscript II (Invitrogen), and sub-jected to real-time PCR with gene-specific primers (seesupplementary material, Table S1) in the presence of 1×Light Cycler 480® probes master mix (Roche). Relativeabundance of mRNA was calculated by normalization toGapdh, using mouse Gapdh endogenous control (LifeTechnologies, 4352339E) or human GAPDH (see sup-plementary material, Table S1).

Luciferase reporter assaysFor the luciferase reporter assay, retrovirally-transducedC2C12 myoblasts or lentivirally-transduced RD cellswere seeded in six-well plates overnight. Luciferasereporters (see supplementary material, Table S2) andTK–Renilla plasmid were co-transfected and 24 h latercells were lysed and luciferase activity assayed usingthe Dual-Luciferase Reporter Assay System (Promega),following the manufacturer’s instructions. All luciferaseactivities were normalized to Renilla activity.

Bioinformatic analysesFor Figure 1E, we determined copy number gainsof the WWTR1 locus (chromosome 3q24-q24) byre-analysing the Innovative Therapies for Childrenwith Cancer/Carte d’Identite ́ des Tumeurs (ITCC/CIT)dataset [33]. For Figures S2A and S4B (see supple-mentary material) the gene expression profile of 235rhabdomyosarcoma patients from two publicly availabledatasets were analysed: the first contained 101 samples(Innovative Therapies for Children with Cancer/Carted’Identite des Tumeurs (ITCC/CIT) [33]; the second134 samples [Children’s Oncology Group/IntergroupRhabdomyosarcoma Study Group (COG/IRSG)] [34].Other non-rhabdomyosarcoma datasets for these figureswere as described in Tremblay et al [13]. For FiguresS2B and S4A (see supplementary material), the CancerCell Line Encyclopaedia [35] search engine was used(http://www.broadinstitute.org/ccle/home/26463/). ForFigure S3A (see supplementary material), WWTR1 andYAP1 mutation and copy number data were plottedusing cBioPortal (http://www.cbioportal.org/) [36,37].For Figure S3B (see supplementary material), selectedsupplementary data from the dataset of Gentles et al[38] were used.

Statistical analysisData were analysed using GraphPad Prism v.5.0 (GraphPad Software) and were presented asmean± standard deviation (SD). Statistical compar-isons were done using one-way ANOVA or unpairedt-test. The Bonferroni multiple comparison test wasapplied following ANOVA to determine significantdifferences between the groups. For all experiments,statistical analysis was conducted on raw data col-lected from at least three independent experiments,performed on different occasions with three replicateseach; p≤ 0.05 was considered significant.

Results

To test whether TAZ protein abundance is associatedwith clinical outcomes in rhabdomyosarcoma, weimmunostained a human rhabdomyosarcoma tissuemicroarray containing 206 ERMS and 76 ARMS casesfor TAZ, using the HPA007415 Sigma antibody, pre-viously used to immunostain breast cancer samples[39], and assessed TAZ levels and location (Figure 1A,C). Nuclear staining for TAZ protein was observedin 22.8% (47/206) of ERMS samples (Figure 1C).Survival curves for TAZ-positive and -negative sampleswere generated for tumours where data were available,and showed lower survival for TAZ-positive rhab-domyosarcomas (Figure 1B). Survival was significantlylower for TAZ-positive ERMS (p= 0.032) and therewas also a trend for lower survival in TAZ-positiveARMS (p= 0.094) when compared to TAZ-negativeERMS and ARMS, respectively. Additionally, wemeasured WWTR1 mRNA levels in human myoblasts[30], RD (ERMS) and RH30 (ARMS) cells [40] byRT–qPCR and also performed a western blot to com-pare TAZ protein levels in these cell lines. RD cellshad the highest WWTR1 mRNA and TAZ protein abun-dance, compared to RH30 cells and human skeletalmyoblasts (Figure 1D). Re-analysis of a previouslypublicly available dataset [33] revealed copy numbergains of the region of Chromosome 3 that incor-porates the WWTR1 locus (chromosome 3q24-q24)in 12% (five of 43) of human fusion gene-negativerhabdomyosarcomas, but not in fusion gene-positiveARMS (Figure 1E). Comparison with expressiondata showed that the expression level of the WWTR1gene correlates with the copy number of its chromo-somal locus, with a Pearson correlation coefficient(r)= 0.31 for fusion gene-negative rhabdomyosarcoma(p= 0.046).

Consistent with the association of TAZ immunos-taining with ERMS rather than ARMS, WWTR1expression was also higher in ERMS than inPAX3/7–FOXO1-positive ARMS and human skeletalmuscle (see supplementary material, Figure S2A).To compare expression of WWTR1 in RD and RH30cells and soft tissue sarcomas generally in relation toother tissue cancer cell lines, we plotted their mRNAlevels using the Cancer Cell Line Encyclopedia dataset(see supplementary material, Figure S2B) [35]. Thisrevealed that WWTR1 and YAP1 (not shown) werehighly expressed in most cancer cell lines, with theexception of low expression in blood cancers. Thisanalysis confirmed that WWTR1 expression was highin ERMS cells consistent with higher expression in theRD (ERMS) than RH30 (ARMS) cell lines (Figure 1D).

We also plotted the pan-cancer mutational profilefor WWTR1 and YAP1 using cBioPortal [36,37]. Addi-tionally, we displayed a meta-z value for WWTR1 andYAP expression as a measure of their association withsurvival in 18 000 cases of human cancer [38] (see sup-plementary material, Figure S3A, B). These analyses


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Figure 1. TAZ is associated with reduced survival in ERMS. (A) Examples of rhabdomyosarcoma, showing either negative or positive TAZimmunostaining; positive TAZ staining shows both strong nuclear and cytoplasmic TAZ staining, shown at higher magnification in FigureS1A (see supplementary material); scale bar= 50 μm. (B) Percentage survival of ERMS and ARMS patients who had sufficient follow-up tobe included in the survival analysis scored positive and negative for TAZ; the table shows patients at each given time point, ie those whohad not died or had been lost to follow-up. (C) Abundance and localization of TAZ in ERMS and ARMS. (D) (Left) Expression of WWTR1and protein abundance in cultured human skeletal myoblasts, RD and RH30 cells; data are presented as mean± SD, where n= 3 and *denotes a significant difference (p< 0.05), as assessed using one-way ANOVA when compared to human myoblasts. (E) Genomic regionson chromosome 3 that include the WWTR1 locus were gained in five of 43 (12%) fusion-negative rhabdomyosarcoma samples [33].

revealed few point mutations in TAZ and YAP1, but somecopy number alterations were found (see supplementarymaterial, Figure S3A). Across all cancers, expressionof TAZ and YAP1 was only moderately associated withpoor survival (see supplementary material, Figure S3B)[38]. BIRC5 and KLRB1 were also plotted as reference

genes whose expression is most associated with poorand good survival, respectively (see supplementarymaterial, Figure S3B).

The positive correlation between high WWTR1expression and poor survival prompted us to testwhether expression of wild-type TAZ or constitutively


TAZ in ERMS 7

Figure 2. TAZ and TAZ S89A enhance proliferation in C2C12 myoblasts. (A) pMSCV map and schematic drawing of the control vector, TAZand TAZ S89A-encoding inserts. (B) Validation of human WWTR1 expression and TAZ protein levels in cells transduced with TAZ or TAZS89A-encoding retroviruses. (C) Example images of C2C12 myoblasts transduced with control vector, TAZ or TAZ S89A-encoding retrovirusesand immunostained 2 days later for the proliferation marker Ki67, and quantification of the proportion of Ki67-expressing myoblasts; scalebar= 50 μm; data are expressed as mean± SD, where n= 3 and * denotes a significant difference (p< 0.05), as assessed using one-wayANOVA when compared to control.

active TAZ S89A was sufficient to transform C2C12mouse myoblasts. We subcloned the pMSCV retroviralbackbone (containing an internal ribosomal entry siteto allow parallel expression of eGFP) with flag-taggedhuman TAZ and TAZ S89A, with pMSCV–IRES–eGFP serving as a control (Figure 2A). Retroviral con-structs were verified by RT–qPCR, where expressionof human WWTR1 mRNA was detected from both theTAZ and TAZ S89A-encoding retroviruses (Figure 2B).Western blot analysis of transduced cells also confirmedthe generation of wild-type TAZ and constitutivelyactive TAZ S89A from the retroviral constructs(Figure 2B). The over-expression of TAZ mutantsin C2C12 myoblasts or knockdown of TAZ in RD cellsdid not affect YAP abundance (Figures 2B, 5A).

To measure the proliferation of C2C12 myoblasts,we transduced myoblasts with the TAZ or TAZ

S89A-encoding retroviruses and immunostained forthe proliferation marker Ki67. Expression of TAZsignificantly increased the proportion of Ki67-positivecells by 59% and TAZ S89A increased it by 96%,compared to control vector (Figure 2C), revealing thatenhanced TAZ levels and activity promotes proliferationin skeletal myoblasts.

Next, we tested the effects of TAZ or TAZ S89Aexpression on C2C12 myoblast colony formationon soft agar, as a measure of transformation. TAZexpression significantly increased the mean number ofcolonies by 12-fold and TAZ S89A by 18-fold whencompared to control vector (Figure 3A). These colonieswere also bigger in size with TAZ or TAZ S89A expres-sion (Figure 3A). TAZ or TAZ S89A expression didnot impair myogenic differentiation in immortalizedC2C12 myoblasts (Figure 3B), consistent with earlier


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Figure 3. TAZ or mutant TAZ S89A promote transformation of C2C12 myoblasts. (A) Example images showing colony formation in C2C12myoblasts transduced with empty vector, TAZ or TAZ S89A-encoding retroviruses; TAZ or TAZ S89A significantly increase colony number andsize when compared to control vector. (B) Expression of TAZ or TAZ S89A did not prevent myogenic differentiation of C2C12 myoblasts whencompared to control (empty vector). TAZ or TAZ S89A, actually significantly increased the fusion index after 72 h. Data are expressed asmean± SD, where n= 3 and * indicates a significant difference (p< 0.05) using one-way ANOVA when compared to control; scale bar= (A)500 μm; (B) 50 μm

findings [22], and in marked contrast to the inhibitoryeffects of YAP1 S127A on differentiation [24].

To test whether the different functions of TAZ andYAP can be explained by different transcriptional activ-ities, we used a panel of Hippo signalling reporters(see supplementary material, Table S2), including the8XGTIIC Hippo [41,42], CTGF (connective tissuegrowth factor; a Hippo reporter with three Tead bindingsites from this gene [32]) and a Brachyury reporter(as YAP has been reported to bind TBX5 in tumourcell lines [8]). C2C12 myoblasts were infected withretroviral vectors encoding wild-type TAZ, TAZ S89Aor YAP1 S127A mutants and then transfected with

reporter constructs. Measuring normalized luciferaseactivity from the reporter constructs revealed thatwhile mutant TAZ S89A or YAP1 S127A activatedthe 8XGTIIC reporter by similar amounts, only YAP1S127A significantly enhanced the activity of the CTGFand Brachyury reporters (Figure 4A).

Next, we searched for transcriptional mechanismsby which TAZ S89A drives tumour-related changes inmyoblasts. To do this we retrovirally expressed TAZor TAZ S89A and used RT–qPCR to measure expres-sion of the YAP activity markers Ctgf , the zebrafishembryonal cancer stem cell gene Myf5 [43] and Phgdh,Psat1 and Psph of the serine biosynthesis pathway,


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Figure 4. TAZ S89A activates genes associated with human cancer. (A) Effects of TAZ, mutant TAZ S89A and YAP S127A expression on Hippopathway reporter constructs 8XGTIIC–Luc, CTGF–Luc and Brachyury–Luc in C2C12 myoblasts. (B) Validation of the expression of humanWWTR1 and effects of TAZ or TAZ S89A expression in C2C12 myoblasts on expression of Ctgf , Myf5, Phgdh, Psat1 and Psph. Data areexpressed as mean± SD, where n= 3 and * indicates a significant difference (p< 0.05) when compared to control using one-way ANOVA

previously linked to remodelling of metabolism in can-cer [44,45] and induced by Yap in myoblasts [23] andYap-driven ERMS [13]. TAZ S89A drove increasedexpression of the cancer-related genes Phgdh, Psat1,Psph and Myf5, although Ctgf mRNA levels wereunchanged (Figure 4B). This effect of TAZ on Myf5in C2C12 myoblasts is consistent with the 8.8-foldup-regulation in YAP1 S127A-driven ERMS [13] andhigh MYF5 expression in the RH18 (ERMS) cell line andin human ERMS compared to skeletal muscle or humanARMS (see supplementary material, Figure S4). Thus,constitutive active TAZ can drive expression of key rhab-domyosarcoma and cancer-related genes.

To test whether TAZ is critical for the tumour char-acteriztics of RD cells, where TAZ protein levelsare high (Figure 1D), we investigated the effects oflentiviral shRNA-mediated TAZ knockdown (see sup-plementary material, Table S3) on the proliferation and

differentiation of RD cells. As expected, RD cellstransduced with TAZ shRNA-encoding lentiviruses hadless TAZ protein and expressed lower levels of TAZmRNA than control cells transduced with scrambledshRNA sequences. Again, manipulation of TAZ pro-tein levels did not alter YAP1 mRNA or YAP proteinlevels (Figure 5A). WWTR1 shRNA-transduced RDcells cultured in soft agar formed significantly fewercolonies when compared to control cells transducedwith scrambled shRNA sequences, which were alsosmaller (Figures 5B). TAZ knockdown in RD cellsalso reduced the proliferation rate, as measured byEdU incorporation (Figure 5C). Immunostaining forMyHC after TAZ knockdown in RD cells revealedthat reduction of TAZ levels did not affect myogenicdifferentiation (Figure 6A).

Next, we performed qPCR analyses to assess theeffect of TAZ knockdown in RD cells on expression


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Figure 5. TAZ knockdown reduces proliferation and transformation behaviour in RD cells. (A) Transduction of RD cells with scrambled orTAZ shRNA-encoding lentiviruses for 24 h followed by 72 h puromycin selection reduced TAZ protein and mRNA but did not affect YAPexpression, as assessed by western blot and qPCR. (B) Example images showing number of colonies and their size with RD cells transducedwith either scrambled or TAZ shRNA; TAZ knockdown significantly reduced colony number when compared to control. (C) Example imagesof RD cells transduced with scrambled or TAZ shRNA lentiviruses and incorporation of EdU visualized and quantified. Data are expressed asmean± SD, where n= 3 and * denotes a significant difference (p< 0.05) when compared to control using unpaired t-test; scale bar= (B)500 μm; (C) 50 μm

of some known Hippo target genes. TAZ knockdownsignificantly reduced the expression of CYR61 by ∼75%and BIRC5 (survivin) by ∼90% when compared to con-trols (Figure 6B). There was also a trend for reducedE2F1 expression after TAZ knockdown, but this didnot reach significance (p= 0.06) (Figure 6B). Further-more, TAZ knockdown in RD cells significantly reducedthe transcriptional activity of the 8XGTIIC (TEAD)luciferase reporter by∼70% (Figure 6B), suggesting thatTEADs are key targets of TAZ in RD cells. Together,these data further indicate that TAZ acts as an oncogenein RD cells.

Discussion

Our data suggest that TAZ functions as an oncogenein ERMS. However, whilst YAP1 S127A expression

blocks myogenic differentiation, we confirm that TAZS89A expression does not inhibit differentiation, inline with an earlier study [24]. Consistent with itsoncogene function, TAZ drives expression of the ERMSstem cell factor Myf5 [43] and of members of theserine biosynthesis pathway (Phgdh, Psat1 and Psph),which have been linked to metabolic remodellingin cancer [44,45]. Moreover, knockdown of TAZ inhuman ERMS RD cells reduces both proliferation andanchorage-independent growth.

WWTR1 mRNA is more highly expressed in ERMSthan in PAX3/7–FOXO1A-positive ARMS and differen-tiated skeletal muscle. Similarly, TAZ protein is detectedin 55% of human ERMS but only in 36% of humanARMS cases. These observations, and our previousresults that identified more YAP immunostaining inERMS than ARMS, suggest that YAP and TAZ aregenerally more abundant in ERMS than in ARMS. Copy


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Figure 6. TAZ knockdown reduces the levels of known oncogenes in RD cells. (A) shRNA-mediated knockdown of TAZ in RD cells had noeffect on myogenic differentiation, as shown by immunostaining with MyHC. (B) Expression of CYR61, E2F1, BIRC5 and activity of the8XGTIIC–Luc reporter was assessed in RD cells after TAZ knockdown. Data are expressed as mean± SD, where n= 3 and * indicates asignificant difference (p< 0.05) when compared to control using unpaired t-test; scale bar= 50 μm

number gains can explain the higher abundance of YAPin some ERMS cases [13,14] and here we additionallyreport copy number gains of the WWTR1 locus in 12%of ERMS and fusion gene-negative ARMS cases, but notin any ARMS case [46].

We previously reported that YAP abundance andactivity are associated with higher grades and reducedsurvival in ERMS [13]. Here we show that the abun-dance of TAZ protein is similarly associated withreduced survival in ERMS (p= 0.032) and that thereis a trend for ARMS (p= 0.094), suggesting that TAZis functionally important, especially in ERMS. Similarassociations between TAZ abundance and poor sur-vival have been reported for human breast cancer [39],non-small cell lung cancer [47], hepatocellular carci-noma [48] and colorectal cancer [49]. In breast cancer,higher Hippo activity was detected in higher-gradebreast cancers and in this cancer TAZ was also linkedto cancer stem cells and drug resistance [39].

Rhabdomyosarcomas comprise cells/myoblast-likecells that fail to differentiate into post-mitotic, multin-ucleated myotubes and fully mature muscle fibres [25].Intriguingly, TAZ expression increases during myogenicdifferentiation of human fetal myoblasts and TAZ hasbeen reported to promote myogenic differentiation [24],whereas YAP inhibits differentiation [20,23]. In ourexperiments, TAZ S89A and TAZ expression increasedproliferation and soft agar growth of C2C12 myoblasts,whereas TAZ knockdown in RD cells reduced prolifera-tion, soft agar growth, activity of the 8XGTIIC reporterand expression of genes such as CYR61 and BIRC5.Collectively, this suggests that TAZ acts, like YAP [13],as an oncogene in the muscle lineage.

Intriguingly, whilst YAP1 S127A (but not YAP)prevents differentiation of C2C12 myoblasts [22], TAZS89A and TAZ enhanced myogenic differentiation intomultinucleated, myosin heavy chain-positive myotubes.This context-dependent, seemingly contradictorypro-proliferation and pro-differentiation function ofTAZ is reminiscent of the function of Notch in themuscle lineage, as Notch can affect both the quiescenceand proliferation of muscle stem cells, depending onthe context and timing [50].

To identify potential mechanisms by which TAZexerts its effects, we conducted reporter and geneexpression experiments. TAZ and YAP mainly bindTEAD transcription factors that regulate gene expres-sion through binding to CATTCC/GGAATG motifs(MCAT; TEADs bind both strands [1,2]). Addition-ally, YAP and TAZ have been reported to bind othertranscription factors, including TBX5 [8,51], whichhas been identified as an important interaction incancer [8]. To identify putative mechanisms for thedifferent actions of YAP and TAZ, we investigatedthe effect of TAZ and YAP mutants on 8XGTIIC,CTGF and Brachyury (T-box) reporters. This revealedthat, whilst TAZ S89A and YAP1 S127A similarlyactivated the 8XGTIIC reporter [41,42], only YAP1S127A was able to significantly activate the CTGF [32]and Brachyury reporters. This suggests that TAZ andYAP have common and unique effects on transcription,which could explain the difference in function seen inskeletal muscle and in Yap1 [15] and Wwtr1 knockoutmice [16].

Finally, we determined the effect of wild-type TAZ orTAZ S89A expression on genes that we had previously


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identified as Yap-regulated genes in myoblasts [23] andYap-driven ERMS [13].

CTGF is commonly used as a marker of Hippo activ-ity, as it has three MCAT Tead-binding sites in its prox-imal promoter [32]. We found no effect of TAZ or TAZS89A expression on Ctgf expression. In contrast to this,YAP S127A increases Ctgf expression in myoblasts[23] but Ctgf expression is not increased in Yap-drivenERMS [13]. This suggests that Ctgf expression is not auniversal marker of high YAP/TAZ-Tead activity.

We found that TAZ and TAZ S89A increased Myf5expression. This is functionally relevant, as Myf5is a cancer stem cell gene in zebrafish ERMS [43].Intriguingly, the ECR111 enhancer of Myf5 harboursa Tead-targeted MCAT motif [52] which could act asa target for TAZ. Previously we found that especiallyYAP1 S127A increased Myf5 expression in C2C12myoblasts and Yap-ERMS [13]. Together this suggeststhat YAP and TAZ may contribute to the maintenanceof a cancer stem cell population in ERMS through theexpression of Myf5.

Cancer cells remodel their metabolism so that gly-colytic intermediates and other metabolites are chan-nelled into biosynthetic pathways to support growthand proliferation. This is known as aerobic glycoly-sis or the Warburg effect [53]. The serine biosynthe-sis pathway, and especially the first enzyme of thispathway, Phgdh, have previously been identified asproliferation-limiting genes in cancer, and some copynumber gains of PHGDH have been identified in somecancers [44,45]. We found that TAZ S89A significantlyincreased expression of Phgdh, Psat1 and Psph, thethree enzymes of the serine biosynthesis pathway. TheTAZ-driven induction of the serine biosynthesis path-way is a novel mechanism of metabolic remodellingand complements previous studies that linked Hippo sig-nalling to the Warburg effect in cancer [54,55].

Interestingly, YAP has recently been shown toinversely regulate TAZ protein, but this inverse rela-tionship is unidirectional, only being observed uponmodulation of YAP and not TAZ [56], This inverserelationship extends to skeletal muscle, since over-expression of TAZ mutants in C2C12 myoblasts orknockdown of TAZ in RD cells do not seem to havecompensatory effects on YAP in RD cells.

In summary, our data identify TAZ as an oncogene inthe muscle lineage. This further supports an importantrole for the Hippo pathway in both ERMS [13], ARMS[57] and sarcoma in general [58,59].

Acknowledgements

This study was funded by Sarcoma UK, Friends ofAnchor and the Medical Research Council grant number99477 awarded to HW and PSZ. This work was also sup-ported, in part, by NHS funding to the NIHR Biomedi-cal Research Centre at the Royal Marsden Hospital andthe Institute of Cancer Research, and the Chris Lucas

Trust, UK. We also thank the CCLG (Children’s cancerand leukaemia group) Tissue Bank for access to sam-ples, and contributing CCLG centres, including mem-bers of the ECMC paediatric network. The CCLG Tis-sue Bank is funded by Cancer Research UK and CCLG.The Zammit lab is also currently supported by MuscularDystrophy UK (RA3-3052), Association Française Con-tre les Myopathies (17865, 20082, 19105 and 16050)and the FSH (Fascioscapulohumeral muscular dystro-phy) Society (Shack Family and Friends research grant(FSHS-82013-06)). In addition, we would like to thankProf Kun-Liang Guan and Prof Malcolm Logan forkindly providing constructs.

Author contributions

Abdalla Mohamed performed most, and Congshan Sunand Vanessa De Mello contributed to, the cell cultureexperiments. Jonanna Selfe and Janet Shipley providedthe tissue microarrays which were immunostained andscored by Abdalla Mohamed and Graeme I Murrayand statistically analysed by Jonanna Selfe and JanetShipley. Edoardo Missiaglia carried out bioinformaticanalyses. Abdalla Mohamed and Henning Wackerhagewrote the manuscript and Peter S Zammit and HenningWackerhage planned the study and obtained funding.

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SUPPLEMENTARY MATERIAL ONLINESupplementary materials and methods

Supplementary figure legends

Figure S1. Examples of immunohistochemistry

Figure S2. Analyses of levels of WWRT1 and YAP1

Figure S3. Mutations in WWTR1 and YAP1, and survival analyses

Figure S4. Analyses of Myf5 expression in cancer cell lines and of MYF5 in different cohorts

Table S1. RT–qPCR primers used in this study

Table S2. Name, description and ratio (firefly:Renilla) of the luciferase constructs used in dual-luciferase reporter assays

Table S3. Catalogue number and the target shRNA sequences of scramble control shRNA and TAZ shRNA


thehippoeffectortaz(wwtr1)transformsmyoblastsandtaz ...€¦ · graeme i murray,1 pete s zammit2...

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